Our long-term goal is to develop novel antitumor therapies to treat recalcitrant non-small cell lung cancers (NSCLCs) by exploiting their unique over-expression of the two-electron, Phase II detoxifying enzyme, NAD(P)H:Quinone Oxidoreductase 1 (NQO1) in combination with inhibitors of poly(ADP-ribose) polymerase inhibitors. In NSCLC, NQO1 is elevated 5- to 200-fold above associated normal tissues, while catalase levels are expressed at extremely lower levels in NSCLCs than in associated normal lung (or all other normal) tissue. We showed that unique and novel NQO1 bioactivatable drugs, (e.g., - lapachone (ARQ761 in clinical trials), are `bioactivated' by NQO1, resulting in massive H2O2-induced, apyrimidinic/apurinic (AP) site- and DNA single-strand break-mediated hyperactivation of poly(ADP- ribosyl) polymerase 1 (PARP1) that causes dramatic NAD+ losses (Huang et al., Cancer Res, 2012). However, co-administration of a PARP inhibitor dramatically enhances the antitumor activities of these drugs (Huang et al., Cancer Cell, 2016). We have discovered a much more potent NQO1 bioactivatable drug, isobutyldeoxynyboquinone (IB-DNQ) that is significantly more synergistic with PARP inhibition that -lapachone. We hypothesize that IB-DNQ will be a significantly better drug alone than ARQ761 (- lapachone) and result in significantly greater synergistic, NQO1-dependent antitumor activity in combination with PARP inhibitors against NQO1+ NSCLC cells. We propose to complete three specific aims to test this hypothesis.: Aim 1: To define the mechanism of action of PARP inhibition + IB-DNQ synergistic cell death (Yrs 0-5); Aim 2: To define the role of IB-DNQ-induced H2O2-related bystander effect in simultaneous NQO1+ programmed necrosis vs NQO1- apoptosis in matched NSCLC cells (Yrs 0-5); and Aim 3: To define the tumor-selective, NQO1+-dependent antitumor effects of the PARP inhibition + IB-DNQ regimen vs IB-DNQ alone in orthotopic NSCLC xenografts (Yrs 0-5). The aims will be performed using genetically matched NQO1+ vs NQO1- NSCLC cell lines by si/shRNA knockdown of NQO1+ human cancer cell lines and/or forced NQO1 over-expression to match known levels in polymorphic NQO1- human NSCLCs. We will also examine genetically match PARP1 si/shRNA knockdown vs normal NSCLC cells in vitro and in vivo. We assembled an outstanding research team to explore the metabolic, bystander, and efficacy effects of PARP inhibition + IB-DNQ treatments in orthotopic NSCLC xenograft models in this 5-yr grant. Our results should lead to the development of a clinical trial using this combination in 5 years.